Caleb Webber

Elevated α-synuclein results in mitochondrial dysfunction, however the exact mechanism is poorly understood. α-Synuclein localises to multiple subcellular sites including mitochondria, where it binds anionic lipids which regulate mitochondrial dynamics through DRP1. We hypothesised that pathogenic levels of α-synuclein lead to dysregulation of mitophagy and lipid-dependent mitochondrial dynamics, causing mitochondrial and neuronal dysfunction. Using induced pluripotent stem cell-dopamine neurons (iPSC-DANs) carrying the α-synuclein gene triplication mutation ( SNCA-Trp ) which causes an early-onset form of Parkinson’s disease (PD), we established that elevated α-synuclein increased basal mitophagy compared to healthy controls and overall loss of mitochondrial mass. This was associated with accumulation of basal mitophagy receptors BNIP3 and NIX and decreased proteasome activity, and was independent of PINK1-Parkin mitophagy. We identified reduced mitochondrial gene expression by single cell RNAseq mirroring mitochondrial depletion. We found increased levels of dynamin-related protein 1 (DRP1)-dependent mitochondrial fission and perturbed levels of anionic lipids, including cardiolipin and phosphatidic acid, two lipids which directly bind to α-synuclein. Pharmacological inhibition of DRP1 reversed the excessive basal mitophagy in SNCA-Trp neurons and restored deficits in cellular ATP and calcium release. Finally, we assessed neuronal function and found that impairment of mitochondrial dysfunction by elevated α-synuclein led to reductions in neuronal firing activity. Inhibition of DRP1 rescued the neuronal activity deficit in SNCA-Trp neurons, suggesting that relieving – instead of increasing – mitophagy pressure may be beneficial in α-synuclein-driven PD.

Pain-related conditions are the leading cause of disability worldwide. Existing GWAS for chronic pain have mainly focused on individual pain-related disorders, which may not optimally capture the phenotype. Here, we define chronic pain based on prescription analgesic use ( ≥ 90 days) in two large biobanks (UK Biobank and FinnGen). GWAS meta-analyses of 11 prescription-based pain phenotypes identify 140 associations with chronic pain, including 78 novel (e.g. ARPP21, CNTNAP2) and 62 previously reported (e.g. SLC39A8, DCC, TRPM8) associations. Integrating these genetic associations with functional data including transcriptome-wide association studies, cell-type and pathway enrichment, and gene enrichment in mouse phenotypes identifies potential mechanisms involved in chronic pain, implicating oligodendrocyte differentiation, neuronal guidance, endolysosomal function and post-synaptic endosome recycling. Our study showcases how the use of prescription data to identify and characterize pain can provide insights into pain genetics and its underlying biology.

The regional specificity of stem cell-derived astrocytes is believed to be an important prerequisite for their application in disease modelling and cell-based therapies. Due to the lack of subtype-defining markers for astrocytes in different regions of the brain, the regional identity of in vitro-derived astrocytes is often declared by the dominant positional characteristics of their antecedent neural progenitors, patterned to a fate of interest, with the assumption that the positional trait is preserved by the derived astrocytes via linear descent. Using a human induced pluripotent stem cell line designed for tracing derivatives of LMX1A ⁺ cells combined with a ventral midbrain induction paradigm, we show that astrocytes originating from LMX1A ⁺ progenitors can only be generated if these progenitors are purified prior to the astrocyte differentiation process, or their progenies are gradually lost to progenies of LMX1A ^- progenitors. This finding indicates that the lineage composition of iPSC-derived astrocytes may not accurately recapitulate the founder progenitor population. Using deep single-cell RNA sequencing, we identified distinct transcriptomic signatures in astrocytes derived from the LMX1A ⁺ progenitor cells. Our study highlights the need for rigorous characterization of pluripotent stem cell-derived regional astrocytes and provides a resource for assessing LMX1A ⁺ ventral midbrain progenitor-derived human astrocytes.

Parkinson’s disease (PD) is a complex neurodegenerative disorder characterised by selective neuronal loss. We integrate deep full-length single-nuclei sequencing of the human substantia nigra with novel genome-wide association studies (GWAS) identifying genetic and cellular drivers of PD. Genetic risk converges on AGTR1+ dopaminergic neurons and perineuronal oligodendrocytes (pODCs), both reduced in PD, as well as oligodendrocyte precursor cells, enriched among disease-disrupted intercellular interactions. AGTR1+ neurons represent a metabolically stressed state, characterised by renin-angiotensin system (RAS) and MAPK activation, oxidative stress, and mitochondrial dysfunction, rather than a distinct subtype. AGTR1+ neurons and pODCs link PD risk to metabolic traits; in pODCs, this association reflects insulin resistance with downregulated PI3K–AKT signalling. GWAS of comorbid PD and type 2 diabetes (T2D) identifies loci in AGTR1 and TCF7L2, while AGTR1+ neurons specifically upregulate RAS and T2D drug targets. Familial PD genes associated to comorbid PD/T2D associate with non-Lewy body PD, stratifying disease mechanisms.

The regional specificity of stem cell-derived astrocytes is believed to be an important prerequisite for their application in disease modelling and cell-based therapies. Due to the lack of subtype-defining markers for astrocytes in different regions of the brain, the regional identity of in vitro-derived astrocytes is often declared by the dominant positional characteristics of their antecedent neural progenitors, patterned to a fate of interest, with the assumption that the positional trait is preserved by the derived astrocytes via linear descent. Using a human induced pluripotent stem cell line designed for tracing derivatives of LMX1A+ cells combined with a ventral midbrain induction paradigm, we show that astrocytes originating from LMX1A+ progenitors can only be generated if these progenitors are purified prior to the astrocyte differentiation process, or their progenies are gradually lost to progenies of LMX1A- progenitors. This finding indicates that the lineage composition of iPSC-derived astrocytes may not accurately recapitulate the founder progenitor population. Using deep single-cell RNA sequencing, we identified distinct transcriptomic signatures in astrocytes derived from the LMX1A+ progenitor cells. Our study highlights the need for rigorous characterization of pluripotent stem cell-derived regional astrocytes, and provides a resource for assessing LMX1A+ ventral midbrain progenitor-derived human astrocytes.

Human cellular models of disease developed using induced pluripotent stem cells (iPSC) have enabled wide-ranging research from investigation of human disease mechanisms to phenotypic drug screens. Pain disorders such as neuropathic pain and headache remain areas of considerable unmet need and are considered high risk by pharma. Human iPSC-derived sensory neurons have already been used to accelerate translational research but the current differentiation protocols produce non-peptidergic nociceptors. We demonstrate for the first time the robust differentiation of hiPSC into peptidergic nociceptor lineage with high yield. These nociceptors express CGRP and TRPV1 whilst the non-peptidergic marker RET was completely absent. Functionally, the nociceptors show functional maturity including the expression of TTX-resistant currents and responding to TRPV1 and TRPA1 agonists. Importantly, they were able to release CGRP basally and upon stimulation by an inflammatory soup, which was inhibited upon the application of the 5-HT _1B/1D/1F agonist, sumatriptan and topiramate, a migraine prophylactic drug. Overall, we report the successful generation of a novel in vitro functional peptidergic nociceptor model which will allow investigation of disease mechanisms in pain and for the development of new effective pain therapies.

Brain perfusion and blood-brain barrier (BBB) integrity are reduced early in Alzheimer’s disease (AD). We performed single nucleus RNA sequencing of vascular cells isolated from AD and non-diseased control brains to characterise pathological transcriptional signatures responsible for this. We show that endothelial cells (EC) are enriched for expression of genes associated with susceptibility to AD. Increased β-amyloid is associated with BBB impairment and a dysfunctional angiogenic response related to a failure of increased pro-angiogenic HIF1A to increased VEGFA signalling to EC. This is associated with vascular inflammatory activation, EC senescence and apoptosis. Our genomic dissection of vascular cell risk gene enrichment provides evidence for a role of EC pathology in AD and suggests that reducing vascular inflammatory activation and restoring effective angiogenesis could reduce vascular dysfunction contributing to the genesis or progression of early AD.

The regional specificity of stem cell-derived astrocytes is believed to be an important prerequisite for their application in disease modelling and cell-based therapies. Due to the lack of subtype-defining markers for astrocytes in different regions of the brain, the regional identity of in vitro -derived astrocytes is often declared by the dominant positional characteristics of their antecedent neural progenitors, patterned to a fate of interest, with the assumption that the positional trait is preserved by the derived astrocytes via linear descent. Using a human induced pluripotent stem cell line designed for tracing derivatives of LMX1A ⁺ cells combined with a ventral midbrain induction paradigm, we show that astrocytes originating from LMX1A ⁺ progenitors can only be generated if these progenitors are purified prior to the astrocyte differentiation process, or their progenies are gradually lost to progenies of LMX1A ^- progenitors. This finding indicates that the lineage composition of iPSC-derived astrocytes may not accurately recapitulate the founder progenitor population. Using deep single-cell RNA sequencing, we identified distinct transcriptomic signatures in astrocytes derived from the LMX1A ⁺ progenitor cells. Our study highlights the need for rigorous characterization of pluripotent stem cell-derived regional astrocytes, and provides a resource for assessing LMX1A ⁺ ventral midbrain progenitor-derived human astrocytes.

Background: Brain perfusion and blood brain barrier (BBB) integrity are reduced in Alzheimer’s disease (AD). We hypothesized that b‐amyloid (Aβ) induces dysfunction of pathways in vascular cells that regulate angiogenesis.Method: We performed single nucleus RNA sequencing (snRNAseq) of vascular cells isolated from post mortem samples from multiple cortical regions from AD patients (Braak V‐VI) and non‐diseased controls (NDC, Braak 0‐II). We developed a meta‐analytic approach to integrate these data with related publicly available datasets. We performed differential gene expression (DGE) analyses as a categorical comparison of AD vs NDC and as a confound‐controlled regression of transcriptomic alterations by regional total Aβ. We also performed cell‐specific gene co‐expression analyses identify modules differentially expressed in AD relative to NDC.Result: Our results describe differential expression of genes central to pathways sustaining angiogenesis, proliferation and migration of endothelial cells and the integrity of the BBB with either AD or greater pathological protein load. HIF1A and FGF2, proangiogenic molecules which both induce VEGF, are upregulated in EC, consistent the parenchymal hypoxic response shown repeatedly in prior studies. However, components of signalling pathways downstream of VEGFR2 are downregulated, suggesting a pathological block to angiogenesis with greater Aβ load. While the proangiogenic stimuli promote the expression of genes such as PPP3CA and ITPR1, coding for calcineurin, which contribute to VEGF signalling through the PLCγ‐Ca²⁺ signalling pathway are upregulated in AD, expression of RAC1 and RHOJ, which enable cellular migration and adhesion, is downregulated. While ANGPT2 expression is also upregulated, the angiopoietin receptor (TIE2, coded by TEK) is downregulated. Downregulation of ADAM10 in AD and upregulation of other NOTCH pathway genes with greater Aβ load suggests altered NOTCH signalling.Conclusion: Overall, a hypoxic tissue environment in AD is accompanied by dysfunctional angiogenesis related to Aβ load. Evidence for multiple sites of dysfunction were identified, involving impaired angiopoietin responses, vascular cell migration and NOTCH signaling.

Advances in stem cell technologies open up new avenues for modelling development and diseases. The success of these pursuits, however, relies on the use of cells most relevant to those targeted by the disease of interest, for example, midbrain dopaminergic neurons for Parkinson’s disease. In the present study, we report the generation of a human induced pluripotent stem cell (iPSC) line capable of purifying and tracing nascent midbrain dopaminergic progenitors and their differentiated progeny via the expression of a Blue Fluorescent Protein (BFP). This was achieved by CRISPR/Cas9-assisted knock-in of BFP and Cre into the safe harbour locus AAVS1 and an early midbrain dopaminergic lineage marker gene LMX1A, respectively. Immunocytochemical analysis and single-cell RNA sequencing of iPSC-derived neural cultures confirm developmental recapitulation of the human fetal midbrain and high-quality midbrain cells. By modelling Parkinson’s disease-related drug toxicity using 1-Methyl-4-phenylpyridinium (MPP⁺), we showed a preferential reduction of BFP⁺ cells, a finding demonstrated independently by cell death assays and single-cell transcriptomic analysis of MPP⁺ treated neural cultures. Together, these results highlight the importance of disease-relevant cell types in stem cell modelling.